DESC:FIELD OF INVENTION:
The present invention relates to an inhalation composition comprising nitazoxanide or its derivatives to a patient (e.g., a human). The present invention further relates to method of treating Coronavirus disease by administering nitazoxanide or derivatives thereof. The present invention also relates to use of the inhalation composition comprising nitazoxanide or its derivatives for the treatment of Coronavirus disease.

BACKGROUND OF INVENTION:
Coronaviruses are a large family of viruses which may cause illness in animals or humans. In humans, several coronaviruses are known to cause respiratory infections ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The most recently discovered coronavirus causes coronavirus disease Coronavirus disease. Coronavirus disease is the infectious disease caused by the most recently discovered coronavirus. The most common symptoms of Coronavirus disease are fever, dry cough, and tiredness. Other symptoms that are less common and may affect some patients include aches and pains, nasal congestion, headache, conjunctivitis, sore throat, diarrhea, loss of taste or smell or a rash on skin or discoloration of fingers or toes. Around 1 out of every 5 people who gets Coronavirus disease becomes seriously ill and develops difficulty breathing. Older people, and those with underlying medical problems like high blood pressure, heart and lung problems, diabetes, or cancer, are at higher risk of developing serious illness. There is multiple ongoing research about numerous antiviral agents, immunotherapies, and vaccines which are being investigated and developed as potential therapies for this serious Coronavirus disease.

Nitazoxanide belongs to the class of drugs known as thiazolides and the class of medications called antiprotozoal agents, and is used to treat diarrhea in children and adults caused by the protozoa Cryptosporidium or Giardia. The antiprotozoal activity of nitazoxanide is believed to be due to interference with the Pyruvate : ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reaction which is essential to anaerobic energy metabolism. Both nitazoxanide and tizoxanide show in vitro activity in inhibiting growth of protozoa. Nitazoxanide is available as tablets and oral suspension. Following oral administration, nitazoxanide is rapidly hydrolyzed to an active metabolite, tizoxanide (desacetyl-nitazoxanide). Tizoxanide then undergoes conjugation, primarily by glucuronidation. Maximum plasma concentrations of the active metabolites tizoxanide and tizoxanide glucuronide are observed within 1-4 hours.

Nitazoxanide has demonstrated superior efficacy against canine coronavirus replication in cell culture assays at low concentration (IC50 1 µg/mL). Recent reports on comparative in vitro efficacy of Nitazoxanide against SARS CoV-2 (Coronavirus disease) in Vero E6 cells at 48 h post infection revealed low EC50 2.12 µM which was comparable with Remdesevir (EC50 0.77 µM) and Chloroquine (EC50 1.13 µM) and moreover, superior to Favipiravir (EC50 61.88 µM), confirming anti SARS-Cov 2 activity in vitro. Further nitazoxanide, and its active metabolite tizoxanide, generally show similar inhibitory activity against viruses in vitro. Both compounds have been shown to inhibit MERS-CoV cultured in LLC-MK2 cells with IC50 values of 0.92 and 0.83 µg/mL for nitazoxanide and tizoxanide, respectively.

Coronavirus disease primarily infects the lungs in the affected individuals and in severe cases causes death due to ARDS and pneumonia. As, Coronavirus disease primarily infects the upper respiratory tract, delivery of drugs via the inhalation route appear to provide a quick and targeted delivery. As therapeutic agents are delivered directly to the lungs, the inhalation route offers a more rapid onset of action, allows smaller doses to be used and has a better efficacy to safety ratio compared to systemic therapy.
US20200276140 discloses a method of treating viral infections by administering niclosamide. The reference discloses a laundry list of active agents including nitazoxanide which could be used in combination with niclosamide, but does not disclose inhalation composition comprising nitazoxanide for use in Coronavirus disease.

There is an unmet need in the art for inhalation composition comprising nitazoxanide or its derivatives thereof for use in Coronavirus disease.

The present invention relates to inhalation composition comprising nitazoxanide or derivatives thereof for use in the treatment of Coronavirus disease. The present invention further relates to method of treating Coronavirus disease by administering nitazoxanide or derivatives thereof. The present invention further relates to inhalation composition comprising tizoxanide for use in treatment of Coronavirus disease.

OBJECT OF THE INVENTION
An object of the present invention is to provide an inhalation composition comprising nitazoxanide or its derivatives to a patient (e.g., a human) for use in treatment of Coronavirus disease.

Another object of the present invention is to provide an inhalation composition comprising nitazoxanide or its derivatives and pharmaceutically acceptable excipients.

One more object of the present invention is a method of treatment of Coronavirus disease by administering nitazoxanide or derivatives thereof.

Yet another object of the present invention is to provide an inhalation composition which is a propellant containing metered dose aerosol comprising nitazoxanide or its derivatives thereof. The aerosol composition may be administered for the treatment of Coronavirus disease.

A further object of the present invention is to provide an inhalation composition which is a nebulization composition comprising nitazoxanide or its derivatives thereof. The nebulization composition may be administered for the treatment of Coronavirus disease.

An object of the present invention is to provide an inhalation composition which is a dry powder composition comprising nitazoxanide or its derivatives thereof. The dry powder inhalation composition may be administered for the treatment of Coronavirus disease.

One more object of the present invention is to provide an inhalation composition which is a nasal spray composition comprising nitazoxanide or its derivatives thereof. The nasal spray composition may be administered for the treatment of Coronavirus disease.

Yet another object of the present invention is to provide an inhalation composition which is a propellant containing metered dose aerosol comprising tizoxanide. The aerosol composition may be administered for the treatment of Coronavirus disease.

A further object of the present invention is to provide an inhalation composition which is a nebulization composition comprising tizoxanide. The nebulization composition may be administered for the treatment of Coronavirus disease.

An object of the present invention is to provide an inhalation composition which is a dry powder composition comprising tizoxanide. The dry powder inhalation composition may be administered for the treatment of Coronavirus disease.
One more object of the present invention is to provide an inhalation composition which is a nasal spray composition comprising tizoxanide. The nasal spray composition may be administered for the treatment of Coronavirus disease.

SUMMARY OF THE INVENTION
The present invention relates to an inhalation composition comprising nitazoxanide or its derivatives to a patient (e.g., a human). The inhalation composition comprises nitazoxanide or its derivatives and pharmaceutically acceptable excipients. The inhalation composition may be a solution, suspension or a powder. The inhalation composition may be filled within a container suitable for inhalation. The inhalation composition may be administered for the treatment of Coronavirus disease. The present invention further relates to method of treating Coronavirus disease by administering nitazoxanide or derivatives thereof.

In one embodiment, the inhalation composition is a propellant containing metered dose aerosol comprising nitazoxanide or its derivatives thereof. The aerosol composition may be administered for the treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof. The nebulization composition may be administered for the treatment of Coronavirus disease.

In one more embodiment, the inhalation composition is a dry powder composition comprising nitazoxanide or its derivatives thereof. The dry powder inhalation composition may be administered for the treatment of Coronavirus disease.

In an embodiment, the inhalation composition is a nasal spray composition comprising nitazoxanide or its derivatives thereof. The nasal spray composition may be administered for the treatment of Coronavirus disease.
In one embodiment, the inhalation composition is a propellant containing metered dose aerosol comprising tizoxanide. The aerosol composition may be administered for the treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a nebulization composition comprising tizoxanide. The nebulziation composition may be administered for the treatment of Coronavirus disease.

In one more embodiment, the inhalation composition is a dry powder composition comprising tizoxanide. The dry powder inhalation composition may be administered for the treatment of Coronavirus disease.

In an embodiment, the inhalation composition is a nasal spray composition comprising tizoxanide. The nasal spray composition may be administered for the treatment of Coronavirus disease.

In one embodiment, the inhalation composition of the present invention is free of preservative.

The inhalation composition may contain about 0.1% w/w to about 40% w/w of nitazoxanide, such as from about 0.2% w/w to about 30% w/w, preferably about 0.5% w/w to about 20% w/w.

The inhalation composition may contain about 0.1% w/w to about 40% w/w of tizoxanide, such as from about 0.2% w/w to about 30% w/w, preferably about 0.5% w/w to about 20% w/w.

In one more embodiment, the inhalation composition of the present invention provided herein has a long shelf life, i.e., it is stable during long term storage. The pharmaceutical composition or solution may contain greater than about 80%, such as greater than about 85%, greater than about 90%, greater than about 95% or greater than about 98% of the initial amount of nitazoxanide or its derivative in the inhalation composition after being stored for 3 or 6 months or 1, 2 or 3 years at 25° C.

In an embodiment, the inhalation composition of the present invention have a pH of about 1 about 9. For example, the preferred pH range of the inhalation composition is about 1 to about 9, preferably about 4 to about 8, more preferably about 5 to about 7.

Another embodiment is a premeasured, prepackaged, premixed inhalation composition. Preferably, the inhalation composition is a ready-to-use composition which does not require any mixing or dilution by the subject prior to administration. The inhalation composition may be administered for the treatment of Coronavirus disease.

Yet another embodiment is a method of administering nitazoxanide or its derivatives thereof by inhalation for use in treatment of Coronavirus disease.

One more embodiment is a kit comprising an inhalation device, instructions for using the device and the container containing the inhalation compositions of the present invention.

Other objects, features and advantages of the present invention will be apparent to those of ordinary skill in the art in view of the following detailed description of the invention and accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an inhalation composition comprising nitazoxanide or its derivatives to a patient (e.g., a human). The inhalation composition comprises nitazoxanide or its derivatives and pharmaceutically acceptable excipients. The inhalation composition may be a solution, suspension or a powder. The inhalation composition may be filled within a container suitable for inhalation. The inhalation composition may be administered for the treatment of Coronavirus disease. The present invention also relates to process of preparing the inhalation compositions.

The term "nitazoxanide or its derivatives thereof” includes pharmaceutically acceptable salts, esters, solvates, hydrates, enantiomers, and polymorphs thereof.

The term "tizoxanide” includes pharmaceutically acceptable salts, esters, solvates, hydrates, enantiomers, and polymorphs thereof.

The term “inhalation” in the present context encompasses administration by inhalation route including oral and nasal inhalation route and includes compositions meant to be administered as dry powder inhalation, metered dose inhalation, nebulization, nasal spray, or insufflations.

Nitazoxanide and Tizoxanide
Nitazoxanide (NTZ), is the generic name for 2- (acetolyloxy) -N- (5-nitro 2-thiazoly) benzamide, a compound first synthesized by Rossignol and Cavier in 1975. The chemical structure for nitazoxanide is as given below:

The preparation and certain uses of nitazoxanide is disclosed in US Patent 3,950,351.
Tizoxanide is a metabolite of nitazoxanide and referred to as desacetyl-nitazoxanide. The IUPAC name for tizoxanide is 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide and the chemical structure is as given below:

The inhalation composition may contain about 0.1% w/w to about 40% w/w of nitazoxanide, such as from about 0.2% w/w to about 30% w/w, preferably about 0.5% w/w to about 20% w/w.

The inhalation composition may contain about 0.1% w/w to about 40% w/w of tizoxanide, such as from about 0.2% w/w to about 30% w/w, preferably about 0.5% w/w to about 20% w/w.

The nitazoxanide or its derivatives may be in micronized form. Suitable micronisation techniques such as dry milling, wet milling, air jet milling, sieving, homogenizing using a homogenizer such as rotor-stator and/or high pressure homogenizer such as a microfluidizer can be used for micronisation of the nitazoxanide or derivatives thereof. Alternately, the nitazoxanide or its derivatives may be in unmicronized form.

Inhalation composition
The inhalation compositions of the present invention are formulated for intranasal or pulmonary delivery. For example, the inhalation compositions may be in the form of solutions, suspensions, drops, inhalation powder. The inhalation compositions may be administered by any conventional means, using a metered dose inhaler (MDI), a dry powder inhaler (DPI), a nebulizer, or a nasal spray device.

The inhalation compositions of the present invention comprise nitazoxanide or its derivatives thereof. Preferably, the inhalation composition of the present invention comprises tizoxanide and pharmaceutically acceptable excipient thereof. Suitable pharmaceutically acceptable excipients may include, but not limited to carrier, a solvent, a thickening agent, propellants, bulking agents, preservatives, a pH regulator, a tonicity agent, a complexing agent, or combinations thereof. The inhalation compositions can be used in the treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a single unit dose composition. In another embodiment, the inhalation composition is a multiple dose composition.

In an embodiment, the inhalation composition of the present invention has a pH of about 1 about 9. For example, the preferred pH range of the inhalation composition is about 1 to about 9, preferably about 4 to about 8, more preferably about 5 to about 7.

The inhalation composition may contain about 0.1% w/w to about 40% w/w of nitazoxanide, such as from about 0.2% w/w to about 30% w/w, preferably about 0.5% w/w to about 20% w/w.

The inhalation composition may contain about 0.1% w/w to about 40% w/w of tizoxanide, such as from about 0.2% w/w to about 30% w/w, preferably about 0.5% w/w to about 20% w/w.

The invention also relates to a kit comprising an inhalation device, instructions for using the device and the container containing the inhalation compositions of the present invention.
The inhalation compositions of the present invention can be administered by a suitable inhalation device.

The term "Geometric Standard Deviation" is the geometric breadth of the best-fitted log-normal function to the particle size data.

The term "Mass Median Aerodynamic Diameter" is the median aerodynamic size of a plurality of particles, typically in a polydisperse population. The "aerodynamic diameter" is the diameter of a unit density sphere having the same settling velocity, generally in air, as a powder and is therefore a useful way to characterize an aerosolized powder or other dispersed particle or particle formulation in terms of its settling behavior. The aerodynamic diameter encompasses particle or particle shape, density, and physical size of the particle or particle. MMAD is determined herein by cascade impaction, unless the context indicates otherwise.

The term "Fine particle dose" is the dose, expressed in µg or the percentage of the total dose, of the aerosolized drug particles with an aerodynamic diameter < 5 micron.

The term "Fine particle fraction" is the ratio of Fine particle dose to the total recovered dose.

The term "D10" is the particle diameter value that 10% of the population of particles lies below.

The term "D50" is the particle diameter value that 50 % of the population lies below and 50% of the population lies above.

The term "D90" is the particle diameter value that 90 % of the population lies below.
The inhalation composition when administered from an inhalation device exhibits a droplet size distribution comprising a D10 of 5-30 microns, a D50 of 20-60 microns, a D90 of 40-150 microns, a SPAN of not more than 5.

The inhalation composition of the present invention exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron.

The inhalation composition of the present invention exhibit a GSD of about 1 to about 5.

The fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Nebulization compositions
The inhalation composition of the present invention may be a nebulization composition. In one embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof and pharmaceutically acceptable excipients. In another embodiment, the inhalation composition is a nebulization composition comprising tizoxanide and pharmaceutically acceptable excipients. In one embodiment, the nitazoxanide or its derivative thereof is in dissolved form. In another embodiment, the nitazoxanide or its derivative thereof is in suspended form.

Suitable pharmaceutical excipients include, but are not limited to stabilizers, complexing agent, isotonic agent, co-solvents or tonicity adjusting agent, pH modifier, buffer, vehicle, and preservatives.

The stabilizers or complexing agents may comprise, but are not limited to, edetic acid (EDTA) or one of the known salts thereof, e.g. sodium EDTA or disodium EDTA dihydrate (sodium edetate), trisodium edetate, edetate calcium disodium edetic acid, citric acid or its hydrate salt, sodium metabisulfite, potassium metabisulfite, ascorbic acid, ascorbyl palmitate, alpha tocopherol, nitrilotriacetic acid, fumaric acid, malic acid, maltol, cyclodextrins such as sulfobutylether-ß-cyclodextrin (SBECD), pentetic acid and the salts or combinations thereof. The nebulization composition may contain from about 1% to about 50% of stabilizer or complexing agent.

Tonicity agents, that may be used, comprise sodium chloride, ammonium carbonate, ammonium chloride, ammonium lactate, ammonium nitrate, ammonium phosphate, ammonium sulfate, ascorbic acid, bismuth sodium tartrate, boric acid, calcium chloride, calcium disodium edetate, calcium gluconate, calcium lactate, citric acid, dextrose, diethanolamine, dimethylsulfoxide, edetate disodium, edetate trisodium monohydrate, fluorescein sodium, fructose, galactose, glycerin, lactic acid, lactose, magnesium chloride, magnesium sulfate, mannitol, polyethylene glycol, potassium acetate, potassium chlorate, potassium chloride, potassium iodide, potassium nitrate, potassium phosphate, potassium sulfate, propylene glycol, silver nitrate, sodium acetate, sodium bicarbonate, sodium biphosphate, sodium bisultite, sodium borate, sodium bromide, sodium cacodylate, sodium carbonate, sodium citrate, sodium iodide, sodium lactate, sodium metabisulfite, sodium nitrate, sodium nitrite, sodium phosphate, sodium propionate, sodium succinate, sodium sulfate, sodium sulfite, sodium tartrate, sodium thiosulfate, sorbitol, sucrose, tartaric acid, triethanolamine, urea, urethan, uridine and zinc sulfate. The tonicity agents are used in the nebulization composition in an amount from about 0.05% to 1.0 % w/w.

The pH may be adjusted by the addition of pharmacologically acceptable acids. Pharmacologically acceptable inorganic acids or organic acids may be used for this purpose. Examples of preferred inorganic acids are selected from the group consisting of hydrochloric acid, hydrobromic acid, nitric acid, sulphuric acid and phosphoric acid. Examples of particularly suitable organic acids are selected from the group consisting of ascorbic acid, citric acid, malic acid, tartaric acid, maleic acid, succinic acid, fumaric acid, acetic acid, formic acid and propionic acid and the like or combinations thereof.

In another embodiment of the present invention, the nebulization composition has a pH from about 2.0 to about 6.0. In another embodiment, the composition has a pH from about 2.0 to about 4.0.

Suitable surfactants that may be used include, but are not limited to, C5-20-fatty alcohols, C5-20-fatty acids, C5-20-fatty acid esters, lecithin, glycerides, propyleneglycol esters, polyoxyethylenes, polysorbates, sorbitan esters and/or carbohydrates. C5-20-fatty acids, propyleneglycol diesters and/or triglycerides and/or sorbitans of the C5-20-fatty acids are preferred, while oleic acid and sorbitan mono-, di- or trioleates are particularly preferred. The nebulization composition may contain about 0% to about 1% surfactant.

In one embodiment, the nebulization composition may be a liposome composition. The liposomal composition may be a ready to use liquid composition or may be a powder composition obtained by a lyophilization or spray drying process. Suitable lipids which may be used include, but are not limited to, neutral lipids, positively-charged lipids, negatively-charged lipids, amphoteric lipids such as phospholipids, and cholesterol are advantageously used. Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine including, but limited to, 1,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limited to, 1,2-dioleylphosphoethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine (DHPE), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), and 1,2-dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated 1,2-diacyl-sn-glycero-3-phosphocholines, 1-acyl-2-acyl-sn-glycero-3-phosphocholines, 1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids. The nebulization composition may contain about 0.01% w/w to about 2%w/w of lipids.

The nebulization composition may optionally include a buffer. General and biological buffers in the pH range of about 2.0 to about 8.0 include, but are not limited to, citric acid/phosphate, acetate, barbital, borate, Britton-Robinson, cacodylate, citrate, collidine, formate, maleate, McIlvaine, phFosphate, Prideaux-Ward, succinate, citrate-phosphate-borate (Teorell-Stanhagen), veronal acetate, MES (2-(N-morpholino)ethanesulfonic acid), BIS-TRIS (bis(2-hydroxyethyl)imino-tris-(hydroxymethyl)methane), ADA (N-(2-acetamido)-2-iminodiacetic acid), ACES (N-(carbamoylmethyl)-2-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3-(N-morpholino)-2-hydroxypropanesulfonic acid), BISTRIS PROPANE (1,3-bis(tris(hydroxymethyl)methylamino)propane), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (N-(2-hydroxyethyl)-piperazine-N'-(2-eth-anesulfonic acid), DIPSO (3-(N,N-bis(2-hydroxyethyl)amino)-2-hydroxypropan-esulfonic acid), MOBS (4-(N-morpholino)-butanesulfonic acid), TAPSO (3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid), TRIZMA® (tris(hydroxymethylaminomethane), HEPPSO (N-(2-hydroxyethyl)piperazine-N'-(2-hydroxy-propanesulfonic acid), POPSO (piperazine-N,N'-bis(2-hydroxypropanesulfonic acid)), TEA (triethanolamine), EPPS (N-(2-hydroxyethylpiperazine-N'-(3-propanesulfon-ic acid), TRICINE (N-tris(hydroxy-methyl)methylglycine), GLY-GLY (glycylglycine), BICINE (N,N-bis(2-hydroxyethyl)glycine), HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)), TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), AMPD (2-amino-2-methyl-1,3-propanediol), and/or any other buffers known to those of skill in the art.

The preservative may comprise one or more of benzalkonium chloride, benzoic acid, benzoates such as sodium benzoate and the like or combinations thereof and such other preservatives which may be known to the person having a skill in the art.

The nebulization compositions of the present invention are formulated with a pharmacologically acceptable vehicle for the dissolution of the nitazoxanide or derivatives thereof to facilitate nebulization and delivery of the actives into the lungs of a patient. Pharmacologically suitable vehicles include, but are not limited to water or alcohols, such as ethanol, isopropanol, and glycols including propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether, glycerol and polyoxyethylene alcohols or combination thereof.

Examples of suitable cosolvents include alcohols, ethers, and glycols. Preferably, the cosolvent is a short chain polar alcohol. More preferably, the cosolvent is an aliphatic alcohol having from one to six carbon atoms, such as ethanol or isopropanol. Examples of suitable ethers include dimethyl ether and diethyl ether. A preferred co-solvent is propylene glycol.

In one embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof, surfactant, complexing agent, isotonicity agent, buffer, optionally a pH adjusting agent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof, Polysorbate 80, disodium EDTA, sodium chloride, citric acid monohydrate and sodium citrate dihydrate, optionally a pH adjusting agent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof, a complexing agent, optionally a pH adjusting agent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof, sulfobutylether-ß-cyclodextrin, optionally a pH adjusting agent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof, surfactant, a cosolvent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof, Polysorbate 80, propylene glycol and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.
In one embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof, surfactant, lipid and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nebulization composition comprising nitazoxanide or its derivatives thereof, Polysorbate 80, Dipalmitoyl phosphatidylcholine and water. In yet another embodiment, the nebulization composition is a powder obtained by spray drying process. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a nebulization liposomal composition comprising nitazoxanide or its derivatives thereof, dipalmitoyl phosphatidylcholine, cholesterol, Polysorbate 80, and water. In yet another embodiment, the nebulization composition is a lyophilized composition. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a nebulization composition comprising tizoxanide, surfactant, complexing agent, isotonicity agent, buffer, optionally a pH adjusting agent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nebulization composition comprising tizoxanide, Polysorbate 80, disodium EDTA, sodium chloride, citric acid monohydrate and sodium citrate dihydrate, optionally a pH adjusting agent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.
In one embodiment, the inhalation composition is a nebulization composition comprising tizoxanide, a complexing agent, optionally a pH adjusting agent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nebulization composition comprising tizoxanide, Betadex Sulfobutyl ether, optionally a pH adjusting agent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a nebulization composition comprising tizoxanide, surfactant, a cosolvent and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nebulization composition comprising tizoxanide, Polysorbate 80, propylene glycol and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a nebulization composition comprising tizoxanide, surfactant, lipid and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nebulization composition comprising tizoxanide, Polysorbate 80, Dipalmitoyl phosphatidylcholine and water. In yet another embodiment, the nebulization composition is a powder obtained by spray drying process. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.
In one embodiment, the inhalation composition is a nebulization liposomal composition comprising tizoxanide, dipalmitoyl phosphatidylcholine, cholesterol, Polysorbate 80, and water. In yet another embodiment, the nebulization composition is a lyophilized composition. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

The present invention will now be explained with reference to the following non-limiting examples.
Example 1
Ingredients Quantity (%w/w)
Nitazoxanide/Tizoxanide 0.5 – 5.0
Polysorbate 80 0.05 – 0.2
Disodium EDTA 0.01 - 0.04
Sodium chloride 0.5 - 0.9
Citric acid monohydrate 0.025 - 0.075
Sodium citrate dihydrate 0.05 - 0.1
Hydrochloric acid 0.1 - 0.5
Water q.s. 5-20 mL

Manufacturing Process:
i. Required quantity of water was taken in a suitable stainless steel container.
ii. Required quantity of Sodium Chloride, Citric acid, sodium citrate & polysorbate 80 were dissolved in water sequentially to form a clear solution.
iii. Nitazoxanide/Tizoxanide was then added to the solution to form a uniform suspension.
iv. pH was adjusted appropriately as required.
v. Final volume was made up by adding water.
vi. The resultant suspension was filled in respules.
Example 2
Ingredients Quantity (%w/w)
Nitazoxanide/Tizoxanide 0.5 – 5.0
Sulfobutylether-ß-cyclodextrin 10 – 75
Hydrochloric acid (for pH adjustment) 1.0
Sodium hydroxide (for pH adjustment) 0.0 – 4.0
Water q.s. to 5-20 mL

Manufacturing Process:
i. Required quantity of water was taken in a suitable stainless steel container.
ii. Required quantity of Sulfobutylether-ß-cyclodextrin was dissolved in water to form a clear solution.
iii. pH of the solution was adjusted to about 6.5
iv. Nitazoxanide/Tizoxanide was then added and dissolved in the solution of step iii to form a clear solution.
v. pH was adjusted appropriately as required.
vi. Final volume was made up by adding water & filtered.
vii. The resultant solution was filled in respules.

Example 3
Ingredient Quantity (%w/w)
Nitazoxanide/Tizoxanide 0.5 – 5.0
Polysorbate 80 0.0-0.02
Propylene glycol 0 – 25
Water for Injection q.s. 3 - 5mL

Manufacturing Process:
i. Nitazoxanide/Tizoxanide was added and dispersed in an aseptic manner in a mixture of Polysorbate 80 and Propylene glycol in Water for Injection in a suitable vessel. Nitrogen flushing was performed throughout the process.
ii. The suspension obtained from step (i) was subjected to size reduction by wet-milling/homogenization to obtain a suspension of desired particle size range.
iii. The suspension was then filled into presterilized depyrogenated glass vials. The vials were stoppered with pre-sterilized stoppers and then sealed.

Example 4
Ingredient Quantity
Nitazoxanide/Tizoxanide 5 - 200 mg
Polysorbate 80 0.001 - 0.2 % w/w
Dipalmitoyl phosphatidylcholine (DPPC) 0.1 - 6.5 mg
Water for Injection q.s. 3 - 5mL

Manufacturing Process:
i. Nitazoxanide/Tizoxanide and Dipalmitoyl Phosphatidyl choline was added and dispersed in an aseptic manner in a mixture of Polysorbate 80 in Water for Injection in a suitable vessel. Nitrogen flushing was performed throughout the process.
ii. The suspension of obtained was subjected to size reduction by wet milling/homogenization to obtain a suspension of desired particle size range.
iii. The suspension was then filled into presterilized, depyrogenated glass vials. The vials were stoppered with pre-sterilized stoppers and then sealed.

Example 5
Ingredient Quantity
Nitazoxanide/Tizoxanide (micronized) 5 - 200 mg
Dipalmitoyl phosphatidylcholine (DPPC) 0.25 – 6.5 mg
Polysorbate 80 0.001 - 0.2 % w/w
Water for Injection q.s. 3 - 5mL

Manufacturing Process:
i. Nitazoxanide/Tizoxanide and Dipalmitoyl Phosphatidyl choline was added and dispersed in an aseptic manner in a mixture of Polysorbate 80 in Water for Injection in a suitable vessel. Nitrogen flushing was performed throughout the process.
ii. The suspension of obtained was subjected to size reduction by homogenization to obtain a suspension of desired particle size range.
iii. The microparticulate suspension obtained in step ii was subjected to spray drying process to remove the solvent.
iv. The dried particles obtained were then filled into presterilized depyrogenated glass vials. The vials were stoppered with pre-sterilized stoppers and then sealed.

Example 6
Ingredient Quantity
Nitazoxanide/Tizoxanide 5 - 200 mg
Cholesterol 1.5- 3.2 mg
Dipalmitoyl phosphatidylcholine (DPPC) 0.5 - 6.5 mg
Chloroform (Removed during process, not present in the final product) q.s.
Methanol (Removed during process, not present in the final product) q.s.
Polysorbate 80 0.80- 0.93 % w/w
water for injection qs 5-10mL
Manufacturing process (aseptic processing)
i. Nitazoxanide/Tizoxanide, Cholesterol and Dipalmitoyl phosphatidylcholine (DPPC) were dissolved in a mixture of chloroform and methanol and passed through 0.2µ filter.
ii. The solvent was then removed by evaporation and a thin film of lipids was obtained.
iii. The lipid membrane was then hydrated with aqueous solution of polysorbate 80 in water for injection.
iv. The resultant suspension was then heated and homogenized to obtain an emulsion.
v. The emulsion was then filled in a suitable vials and sealed.

Example 7
Ingredient Quantity
Nitazoxanide/Tizoxanide 5 - 200 mg
Cholesterol 1.5- 3.2 mg
Dipalmitoyl phosphatidylcholine (DPPC) 0.5 - 6.5 mg
Chloroform (Removed during process, not present in the final product) q.s.
Methanol (Removed during process, not present in the final product) q.s.
Polysorbate 80 0.80- 0.93 % w/w
water for injection qs (5-10mL)

Manufacturing process (aseptic processing)
i. Nitazoxanide/Tizoxanide, Cholesterol and Dipalmitoyl phosphatidylcholine (DPPC) was dissolved in a mixture of chloroform and methanol and passed through 0.2µ filter.
ii. The solvent was then removed by evaporation and a thin film of lipids was obtained.
iii. The lipid membrane was then hydrated with aqueous solution of polysorbate 80 in water for injection.
iv. The resultant suspension was then heated and homogenized to obtain an emulsion.
v. The emulsion was then filled in suitable vials, partially sealed and subjected to a lyophilization process to obtain a dry power. The vials were then sealed.
The nebulization composition can be administered by suitable devices commonly known in the art.

In one embodiment, the inhalation composition when administered from an inhalation device exhibits a droplet size distribution comprising a D10 of 5-30 microns, a D50 of 20-60 microns, a D90 of 40-150 microns, a SPAN of not more than 5. In another embodiment, the inhalation composition of the present invention exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron. In a further embodiment, the inhalation composition of the present invention exhibit a GSD of about 1 to about 5. In a further embodiment, the fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Dry powder inhalation compositions
The inhalation composition of the present invention may be a dry powder inhalation composition. In one embodiment, the inhalation composition is a dry powder inhalation composition comprising nitazoxanide or its derivatives thereof and pharmaceutically acceptable excipients. In another embodiment, the inhalation composition is a dry powder inhalation composition comprising tizoxanide and pharmaceutically acceptable excipients.
In one embodiment, the dry powder inhalation composition is a single unit dose composition. Such a composition may be packaged into capsules. In another embodiment, the dry powder inhalation composition is a multiple dose composition. Such a composition may be packaged into blisters.

Example of a suitable excipient includes a bulking agent. Such bulking agents may include, but are not limited to, lactose, mannitol, trehalose, raffinose, amino acids such as L-leucine and maltodextrins. In some embodiments, it may be desirable to include a bulking agent to improve the physical stability of the composition. Furthermore, in some embodiments, the bulking agent may also improve the chemical stability of the inhalation composition. Other additives known to those of ordinary skill in the art may also be included. Generally, excipients suitable for use may be included in an amount of about 80% or less by weight of the composition, about 50% or less by weight of the composition, 30% or less by weight of the composition, or 10% or less by weight of the composition.

The dry powder compositions may further comprise pharmaceutically acceptable excipients, including, without limitation, pH adjusting and buffering agents and/or tonicity adjusting agents, such as, for example, sodium chloride, potassium chloride, calcium chloride, sodium acetate, sodium lactate, etc.

In one embodiment, the inhalation composition is a dry powder inhalation composition comprising nitazoxanide or its derivatives thereof, and a bulking agent. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a dry powder inhalation composition comprising nitazoxanide or its derivatives thereof, lactose and L-leucine. In yet another embodiment, the dry powder inhalation composition is prepared by spray drying. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a dry powder inhalation composition comprising tizoxanide, and a bulking agent. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a dry powder inhalation composition comprising tizoxanide, lactose and L-leucine. In yet another embodiment, the dry powder inhalation composition is prepared by spray drying. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

The present invention will now be explained with reference to the following non-limiting examples.
Ingredients Quantity (%w/w)
Nitazoxanide/Tizoxanide 5 – 20
L-Leucine 0.1 – 5
Lactose 50 – 95
Acetonitrile q.s
Water q.s
Manufacturing Process:
i. Required quantity of Acetonitrile & Water were mixed in suitable stainless steel container.
ii. L-Leucine & Nitazoxanide/Tizoxanide were dissolved in the above solution sequentially to form a clear solution.
iii. The solution was spray dried in a spray dryer at specified conditions.
iv. The resultant powder was blended with specified quantity of lactose.
v. The final powder was filled in suitable size HPMC capsules

The dry powder inhalation composition can be administered by suitable devices commonly known in the art.

In one embodiment, the inhalation composition when administered from an inhalation device exhibits a MMAD of below about 10 micron, between about 2 micron to 10 micron. In a further embodiment, the inhalation composition of the present invention exhibit a GSD of about 1 to about 5. In a further embodiment, the fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Propellant containing metered dose aerosols
The inhalation composition of the present invention may be a metered dose inhalation composition. In one embodiment, the inhalation composition is a metered dose inhalation composition comprising nitazoxanide or its derivatives thereof and pharmaceutically acceptable excipients. In another embodiment, the inhalation composition is a metered dose inhalation composition comprising tizoxanide and pharmaceutically acceptable excipients.

Suitable pharmaceutical excipients include, but are not limited to propellant, a co- solvent, a surfactant, a buffer/pH adjusting agent, a preservative, a complexing agent, antioxidants or combinations thereof.

Suitable propellants include hydrocarbons such as n-propane, n-butane or isobutane or mixtures of two or more such hydrocarbons such as monofluorotrichloromethane, dichlorodifluoromethane and halogen-substituted hydrocarbons, for example fluorine-substituted methanes, ethanes, propanes, butanes, cyclopropanes or cyclobutanes, particularly 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA227) or mixtures of two or more such halogen-substituted hydrocarbons. In one embodiment, the nitazoxanide or its derivative thereof is in dissolved form. In another embodiment, the nitazoxanide or its derivative thereof is in suspended form.

Examples of suitable cosolvents include alcohols, ethers, and glycols. Preferably, the cosolvent is a short chain polar alcohol. More preferably, the cosolvent is an aliphatic alcohol having from one to six carbon atoms, such as ethanol or isopropanol. Examples of suitable ethers include dimethyl ether and diethyl ether. The metered dose inhalation composition may contain about 0.1% w/w to about 10%w/w of co-solvents.

The surfactants may be selected from oils such as corn oil, olive oil, cottonseed oil & sunflower oil, mineral oil like liquid paraffin, oleic acid, phospholipids such as lecithin and citric acid, sorbitan trioleate, glycerol, glycol, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyethylene glycol, propoxyiated polyethylene glycol, polyoxyethylene lauryl ether, and purified diethylene glycol monoethyl ether. The metered dose inhalation composition may contain about 0.1% w/w to about 10% w/w of surfactant.

Suitable buffers include, but not limited to, acetate, citrate, glutamate, phosphate, benzoate, lactate, ascorbate, tartarate, succinate, glycine, triethanolamine, diethanolamine, tromethamine or suitable mixture thereof.

The inhalation composition of the present invention may contain a preservative. Suitable preservatives include, but not limited to benzalkonium chloride or benzoates such as sodium benzoate, sorbic acid or sorbates such as potassium sorbates and the like.
Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof..

Suitable antioxidants which may be used in the inhalation compositions of the invention, include, but are not limited to, glycine, a-tocopherol, a-tocopherol Polyethylene Glycol Succinate (Vitamin E TPGS), ascorbic acid, propyl gallate, Butylated Hydroxy Anisole (BHA), Butylated Hydroxy Toluene (BHT),

In one embodiment, the inhalation composition is a metered dose inhalation composition comprising nitazoxanide or its derivatives thereof, a propellant, a co-solvent, and optionally a surfactant. The inhalation composition is used in the treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a metered dose inhalation composition comprising nitazoxanide or its derivatives thereof, HFA-134a (1,1,1,2-tetrafluoroethane), and ethanol. The inhalation composition is used in the treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a metered dose inhalation composition comprising tizoxanide, a propellant, a co-solvent, and optionally a surfactant. The inhalation composition is used in the treatment of Coronavirus disease.

In one more embodiment, the inhalation composition is a metered dose inhalation composition comprising tizoxanide, HFA-134a (1,1,1,2-tetrafluoroethane), and ethanol. The inhalation composition is used in the treatment of Coronavirus disease.

The metered dose inhalation composition can be administered by suitable devices commonly known in the art.

In one embodiment, the inhalation composition when administered from an inhalation device exhibits a droplet size distribution comprising a D10 of 5-30 microns, a D50 of 20-60 microns, a D90 of 40-150 microns, a SPAN of not more than 5. In another embodiment, the inhalation composition of the present invention exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron. In a further embodiment, the inhalation composition of the present invention exhibit a GSD of about 1 to about 5. In a further embodiment, the fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Nasal spray compositions
The inhalation composition of the present invention may be a nasal spray composition. In one embodiment, the inhalation composition is a nasal spray composition comprising nitazoxanide or its derivatives thereof and pharmaceutically acceptable excipients. In another embodiment, the inhalation composition is a nasal spray composition comprising tizoxanide and pharmaceutically acceptable excipients. In one embodiment, the nitazoxanide or its derivative thereof is in dissolved form. In another embodiment, the nitazoxanide or its derivative thereof is in suspended form.

Suitable pharmaceutical excipients include, but are not limited to vehicle, preservative, suspending agent, wetting agent, complexing agent tonicity agent.

Pharmacologically suitable vehicle for use herein include, but are not limited to, polar solvents, or compounds that contain hydroxyl groups or other polar groups. Solvents include water or alcohols, such as ethanol, isopropanol, and glycols including propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether, glycerol and polyoxyethylene alcohols. Polar solvents also include protic solvents, including, but not limited to, water, aqueous saline solutions with one or more pharmaceutically acceptable salt(s), alcohols, glycols or a mixture thereof.

The nasal spray composition may optionally include a buffer. General and biological buffers in the pH range of about 2.0 to about 8.0 include, but are not limited to, citric acid/phosphate, acetate, barbital, borate, Britton-Robinson, cacodylate, citrate, collidine, formate, maleate, McIlvaine, phFosphate, Prideaux-Ward, succinate, citrate-phosphate-borate (Teorell-Stanhagen), veronal acetate, MES (2-(N-morpholino)ethanesulfonic acid), BIS-TRIS (bis(2-hydroxyethyl)imino-tris-(hydroxymethyl)methane), ADA (N-(2-acetamido)-2-iminodiacetic acid), ACES (N-(carbamoylmethyl)-2-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3-(N-morpholino)-2-hydroxypropanesulfonic acid), BISTRIS PROPANE (1,3-bis(tris(hydroxymethyl)methylamino)propane), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (N-(2-hydroxyethyl)-piperazine-N'-(2-eth-anesulfonic acid), DIPSO (3-(N,N-bis(2-hydroxyethyl)amino)-2-hydroxypropan-esulfonic acid), MOBS (4-(N-morpholino)-butanesulfonic acid), TAPSO (3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid), TRIZMA® (tris(hydroxymethylaminomethane), HEPPSO (N-(2-hydroxyethyl)piperazine-N'-(2-hydroxy-propanesulfonic acid), POPSO (piperazine-N,N'-bis(2-hydroxypropanesulfonic acid)), TEA (triethanolamine), EPPS (N-(2-hydroxyethylpiperazine-N'-(3-propanesulfon-ic acid), TRICINE (N-tris(hydroxy-methyl)methylglycine), GLY-GLY (glycylglycine), BICINE (N,N-bis(2-hydroxyethyl)glycine), HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)), TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), AMPD (2-amino-2-methyl-1,3-propanediol), and/or any other buffers known to those of skill in the art.
Preservatives include, but are not limited to, phenylethyl alcohol, benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate and phenylethyl alcohol.

Suspending agents which can be used in the nasal spray composition of the present invention include, but are not limited to polyoxyethylene sorbitan fatty esters or polysorbates, including, but not limited to, polyethylene sorbitan monooleate (Polysorbate 80), polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 65 (polyoxyethylene (20) sorbitan tristearate), polyoxyethylene (20) sorbitan mono-oleate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate; lecithins; alginic acid; sodium alginate; potassium alginate; ammonium alginate; calcium alginate; propane-1,2-diol alginate; agar; carrageenan; locust bean gum; guar gum; tragacanth; acacia; xanthan gum; karaya gum; pectin; amidated pectin; ammonium phosphatides; microcrystalline cellulose; methylcellulose; hydroxypropylcellulose; hydroxypropylmethylcellulose; ethylmethylcellulose; carboxymethylcellulose; sodium, potassium and calcium salts of fatty acids; mono-and di-glycerides of fatty acids; acetic acid esters of mono- and di-glycerides of fatty acids; lactic acid esters of mono-and di-glycerides of fatty acids; citric acid esters of mono-and di-glycerides of fatty acids; tartaric acid esters of mono-and di-glycerides of fatty acids; mono-and diacetyltartaric acid esters of mono-and di-glycerides of fatty acids; mixed acetic and tartaric acid esters of mono-and di-glycerides of fatty acids; sucrose esters of fatty acids; sucroglycerides; polyglycerol esters of fatty acids; polyglycerol esters of polycondensed fatty acids of castor oil; propane-1,2-diol esters of fatty acids; sodium stearoyl-21actylate; calcium stearoyl-2-lactylate; stearoyl tartrate; sorbitan monostearate; sorbitan tristearate; sorbitan monolaurate; sorbitan monooleate; sorbitan monopalmitate; extract of quillaia; polyglycerol esters of dimerised fatty acids of soya bean oil; oxidatively polymerised soya bean oil; and pectin extract. In some embodiments, the nasal spray formulations comprise polysorbate 80, microcrystalline cellulose, carboxymethylcellulose sodium and/or dextrose. The nasal spray composition may contain about 0.01% w/w to about 5% of suspending agent.

Suitable surfactants that may be used include, but are not limited to, C5-20-fatty alcohols, C5-20-fatty acids, C5-20-fatty acid esters, lecithin, glycerides, propyleneglycol esters, polyoxyethylenes, polysorbates, sorbitan esters and/or carbohydrates. C5-20-fatty acids, propyleneglycol diesters and/or triglycerides and/or sorbitans of the C5-20-fatty acids are preferred, while oleic acid and sorbitan mono-, di- or trioleates are particularly preferred. The nasal spray composition may contain about 0% to about 1% surfactant.

Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof. The nasal spray composition may contain about 0.0005%w/w to about 0.5% w/w of complexing agent.

Tonicity agents, that may be used, comprise sodium chloride, ammonium carbonate, ammonium chloride, ammonium lactate, ammonium nitrate, ammonium phosphate, ammonium sulfate, ascorbic acid, bismuth sodium tartrate, boric acid, calcium chloride, calcium disodium edetate, calcium gluconate, calcium lactate, citric acid, dextrose, diethanolamine, dimethylsulfoxide, edetate disodium, edetate trisodium monohydrate, fluorescein sodium, fructose, galactose, glycerin, lactic acid, lactose, magnesium chloride, magnesium sulfate, mannitol, polyethylene glycol, potassium acetate, potassium chlorate, potassium chloride, potassium iodide, potassium nitrate, potassium phosphate, potassium sulfate, propylene glycol, silver nitrate, sodium acetate, sodium bicarbonate, sodium biphosphate, sodium bisultite, sodium borate, sodium bromide, sodium cacodylate, sodium carbonate, sodium citrate, sodium iodide, sodium lactate, sodium metabisulfite, sodium nitrate, sodium nitrite, sodium phosphate, sodium propionate, sodium succinate, sodium sulfate, sodium sulfite, sodium tartrate, sodium thiosulfate, sorbitol, sucrose, tartaric acid, triethanolamine, urea, urethan, uridine and zinc sulfate.

In one embodiment, the inhalation composition is a nasal spray composition comprising nitazoxanide or its derivatives thereof, suspending agent, preservative, tonicity adjusting agent, complexing agent, buffer, surfactant and water. The inhalation composition is used in the treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nasal spray composition comprising nitazoxanide or its derivatives thereof, xanthan gum, benzalkonium chloride, sodium chloride, disodium EDTA, phosphate buffer, Polysorbate 80 and water. The inhalation composition is used in the treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a nasal spray composition comprising tizoxanide, suspending agent, preservative, tonicity adjusting agent, complexing agent, buffer, surfactant and water. The inhalation composition is used in the treatment of Coronavirus disease.

In one more embodiment, the inhalation composition is a nasal spray composition comprising tizoxanide, xanthan gum, benzalkonium chloride, sodium chloride, disodium EDTA, phosphate buffer, Polysorbate 80 and water. The inhalation composition is used in the treatment of Coronavirus disease.

The nasal spray composition can be administered by suitable devices commonly known in the art.

In one embodiment, the inhalation composition when administered from an inhalation device exhibits a droplet size distribution comprising a D10 of 5-30 microns, a D50 of 20-60 microns, a D90 of 40-150 microns, a SPAN of not more than 5. In another embodiment, the inhalation composition of the present invention exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron. In a further embodiment, the inhalation composition of the present invention exhibit a GSD of about 1 to about 5. In a further embodiment, the fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Method of treatment
The inhalation compositions comprising nitazoxanide or derivatives thereof cam be used for the treatment of Coronavirus disease. The present invention further relates to method of treating Coronavirus disease by administering nitazoxanide or derivatives thereof. In one embodiment, the inhalation composition is a metered dose composition. In another embodiment, the inhalation composition is a dry powder composition. In one more embodiment, the inhalation composition is a nebulization composition. In a further embodiment, the inhalation composition is a nasal spray composition.

The inhalation compositions of the present invention comprising tizoxanide can be used for the treatment of Coronavirus disease. In one embodiment, the inhalation composition is a metered dose composition. In another embodiment, the inhalation composition is a dry powder composition. In one more embodiment, the inhalation composition is a nebulization composition. In a further embodiment, the inhalation composition is a nasal spray composition.

Nitazoxanide and Tizoxanide have been evaluated for In vitro cytotoxicity studies, permeability studies as well as the cellular uptake studies, as given below:

(A). In vitro cytotoxicity studies for Nitazoxanide, Tizoxanide, in human nasal epithelial (RPMI2650) cells and human alveolar epithelial (A549) cells
Material and Method
Reagents
Cell line: A549
Cell type: Lung epithelial cells
Reviving Passage no: 44
Media: MEM Alpha media with 10% FBS (complete media)

Cell line: RPMI 2650
Cell type: Nasal epithelial cell
Reviving Passage no: 20
Media: MEM Alpha media with 10% FBS

Identification: MEM Alpha media
Supplier: Gibco
Batch No.: 2217403
Physical Description: Pink colored solution
Storage Conditions: 4-8oC

Identification: Fetal Bovine serum
Supplier: Gibco
Batch No.: 42F1383K
Physical Description: Yellowish brown solution
Storage Conditions: 4-8oC

Identification: Trypsin
Supplier: Himedia
Batch No.: 0000381648
Physical Description: Pink colored solution
Storage Conditions: 4-8oC

Identification: Antibiotic Antimycotic solution
Supplier: Himedia
Batch No.: 0000360171
Physical Description: Colourless liquid
Storage Conditions: 4-8oC

Methods:
Cell proliferation assay- A549 and RPMI2650 cells were seeded in 96-well plate at a cell density of 0.5x106 cells per plate. After seeding the plates were cultured overnight at 37 °C, 5 % CO2. 24hr post seeding cytotoxicity was assessed by using seven concentrations starting from 50, 30, 10, 3, 1, 0.3 and 0.1 µMol/L for Nitazoxanide and Tizoxanide and were plated in triplicate. Cells were incubated for 24h and 48h timepoint. At the end of incubation period, MTS reagent was added (20 µL per well) and absorbance was measured at 460 nm. Data was acquired using Spectramax microplate reader (Molecular devices, CA, USA). Data was analysed to determine the % proliferation rate at each time point and concentration tested.
Stock preparation:
100 mM stock was prepared in DMSO, as shown below in table.1. Working stock was prepared in complete media. 10X working stocks are prepared and serial dilutions is performed. 20 ul/well of each 10x working stock was added in respective wells giving the desired concentrations, as mention in the method section.
Compounds MW DMSO stock wt (g) DMSO Vol (ml)
Nitazoxamide 307.28 100mM 15.2146 0.495
Tizoxanide 265.25 100 mM 10.5108 0.396

Results:
In alveolar derived epithelium cells, Tizoxanide and Nitazoxanide showed 75% proliferation and 25-27% cytotoxicity at the highest test concentration of 50µM with no time dependant increase. Remarkably, in nasal derived cell line RPMI 2650 Nitazoxanide and Tizoxanide showed less cytotoxic effect. Tizoxanide and Nitazoxanide showed 100% proliferation and merely 6% cytotoxicity at the highest test concentration with Nitazoxanide at 4h ime point.

Proliferation assay:
% proliferation observed in A549 treated with Nitazoxanide and Tizoxanide
Tizoxanide Nitazoxanide
conc. 24h 48h conc. 24h 48h
50µM 75 72 50µM 75 71
30 µM 109 79 30 µM 116 81
10 µM 101 85 10 µM 100 94
3 µM 98 82 3 µM 105 98
1 µM 97 81 1 µM 105 95
0.3 µM 100 83 0.3 µM 102 96
0.1 µM 100 92 0.1 µM 100 98

% proliferation observed in RPMI2650 treated with Nitazoxanide and Tizoxanide

Tizoxanide Nitazoxanide
conc. 24h 48h conc. 24h 48h
50µM 111 100 50µM 112 94
30 µM 112 103 30 µM 111 101
10 µM 110 108 10 µM 110 107
3 µM 110 114 3 µM 109 105
1 µM 111 109 1 µM 110 107
0.3 µM 110 108 0.3 µM 110 105
0.1 µM 110 108 0.1 µM 111 107

Using the alveolar and nasal derived epithelial cells, Tizoxanide and Nitazoxanide showed close to 20% cytotoxicity at the highest test concentration of 50µM which was way high concentration then the EC90 for SARS-CoV-2 , as identified at 4.64 µM. Thus higher safety window for using higher concentration that can easily affect the virus clinically. Thus the tested concentration is much higher than the effect dose of the drug which are safe enough to be effective, to be use in vitro studies. This itself will validate the use of these drugs which will have increase bioavailability which will tested in-house.

(B) Permeability study using bronchial epithelail cells or nasal derived cells:

Cell line: A549, Calu-3 and RPMI 2650
Cell density: 2.5 x 105 cells/mL per well (n=2/ concentration)
Compounds: Nitazoxanide/ Tizoxanide
Drug concentration: µM and - µM
Incubation: 0 min, 5min, 15 min, 30 min, 45min, 60min, 90min & 120min

1. Alveolar and bronchial epithelial cell lines were cultured in freshly prepared MEM media containing NEAA along with 1 mM sodium pyruvate, and 2 mM L-Glutamine. For 50 mL of complete culture media, 5 mL of filter sterile fetal bovine serum, 100 µL Antibiotic solution in 45 mL MEM and filter sterilized using 0.2-micron filter and sterile 20 mL syringe was added.
2. Cells were seeded using 6-well transwell culture insert having a surface area of 4.5 cm2, at the density of 2x105 cells/ml.
3. 1 ml cell suspension containing 2x105 cells/ml to 2.5x105 cells/ml was seeded to the apical (A) side of the hanging cell culture inserts and 2 ml of complete media is added to all the basal (B) wells. For a 24 well culture inserts (having a surface area of 0.3 cm2) 400 µL of cell suspension was seeded to the apical and 800 µL of complete media to all the basal wells.
4. Plates were incubated and maintained at 37 °C, 5 % CO2 till the day of the assay (7 - 12 days) with alternate day media change, from the basal side post 24 hours of seeding while apical side is kept empty.
5. On the day of the assay, fresh buffer was prepared as follows: Modified HBSS buffer with 10 mM HEPES (i.e. 1191.1 mg of HEPES in 500 mL of commercially available HBSS), pH to 7.4 (± 0.05).
6. Monolayer integrity was tested by using the Trans epithelial electrical resistance (TEER) before initiating the assay by immersing electrodes in each well of the 6 well or 24 well plate so that the shorter arm is dipped into the apical side and the longer arm into the basal side of the well.
7. Wells with TEER values = 300 ohms.cm2 are used for the assay. TEER values of bronchial epithelial cells, in general, are achieved above 300 ?*cm2 from day 10 and sustain at 400 ?*cm2 till day 12. Thus, the assay is scheduled in between day 7 to day 12 post seeding.
8. Unidirectional assay (direct drug dosing) is performed after taking the TEER readings.
9. For 6 well culture inserts 1 ml of HBSS buffer is added to the apical side and 2 ml to the basal side of the wells or add 400 µL of HBSS buffer to the apical side of the wells and 800 µL to the basal side if 24 well cell culture inserts are used.
10. 1ml or 450 µL of the desired concentrations of the test compounds (Nitazoxanide and Tizoxanide) in HBSS buffer (pH 7.4 ) were added to the respective assigned wells while 2 ml or 800 µL of HBSS buffer to the basal side of all the wells to initiate the assay.
11. 50 µL sample from the apical side as a 0 min sample (A0) was collected as a mass balance sample This is replaced with 50 µL of HBSS buffer (pH 7.4) followed which the plate was transferred on an incubator shaker set at 70-90 rpm at 37°C in between each timepoints and sample collection.
12. In following time points, 100 µL samples from the basal side at different time points was collected till the final sample collection timepoint is reached, eg. 120 min. At every time point after collecting the sample the well was immediately replenished with 100 µL of HBSS buffer.
13. After collecting the last time point sample, 50 µL sample from the apical side (A120) of the wells was collected as a mass balance sample.
14. At the end of the assay, TEER value is re-captured to confirm if the cell layer integrity is retained throughout the course of the experiment along with Sodium Fluorescence (Na Flu) permeability.
15. The test drug (Nitazoxanide and Tizoxanide) will be considered as a high permeable or low permeable depending upon the Papp calculated, using the formula as mentioned. Papp formula: Papp = (dQ/dt)*VR/(A*C0) Where, dQ/dt is the cumulative amount in the receiver compartment versus time in µmoles/s, VR is the volume in receiver well, A is the area of the cell monolayer, C0 is the initial concentration of the dosing solution.
The permeability data with an Papp value range above 200 nm/s indicating high permeability for the drug using Caco-2 cells can be implicit, thus validating the use of these drugs with increase bioavailability for elucidating the therapeutic effect against the coronavirus.

(C) Intracellular cellular uptake assay
Cell line: A549 and RPMI 2650
Cell density: 0.5 million per well
Compounds: Nitazoxanide/ Tizoxanide
Drug concentration: µM and - µM
Incubation: 15 min, 30 min, 1hr, 2hr & 4hr post addition for all the timepoints. The plates to be incubated at 37°C, 5% CO2 incubator.

Study protocol:
a) A549 and RPMI 2650 cells were seeded in a 6-well plate at a density of 0.5-1 x 106 cells / mL. Post seeding the cells were incubated overnight at 370C, 5% CO2.
b) After incubation, media from the wells was removed and replaced with fresh media containing various concentrations of Nitazoxanide and Tizoxanide.
c) The cells were incubated with the drugs for different time points raining from 15 mins to 24 hrs.
d) After incubation the supernatant from individual wells was collected in 2 mL tubes and centrifuged. After centrifugation the supernatant collected and stored till quantification for determining the extracellular drug concentrtion.
e) For intracellular levels detection, cells were scraped using a cell scraper from all wells. This cell suspension from individual wells were collected in labelled 2 mL tubes and centrifuged at 500g for 10 mins.
f) Supernatant was discarded and 500 µL of ice cold acidic methanol was added to the pellet obtained in all the tubes followed by incubation for 10-15 minutes for cell lysis.
g) At the end of the incubation time, the tubes were centrifuged at 500g for 20 mins, supernatant was colected and stored at - 80°C., till further analysis
h) The above procedure was repeated for all the mentioned timepoints.

Based on our preliminary invitro data it is clearly suggestive that the working concentration is likely to be achieve the intracellular levels, which are therapeutic concentration that are inhibitory for virus infection and efficiently sensitive towards coronavirus.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and application of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as described.

,CLAIMS:
1. An inhalation composition comprising nitazoxanide or its derivatives for use in treatment of Coronavirus disease.
2. The inhalation composition according to claim 1, wherein the nitazoxanide or its derivative is tizoxanide.
3. The inhalation composition according to claim 1, wherein the inhalation composition is a propellant containing metered dose aerosol.
4. The inhalation composition according to claim 1, wherein the inhalation composition is a nebulization composition.
5. The inhalation composition according to claim 1, wherein the inhalation composition is a dry powder composition.
6. The inhalation composition according to claim 1, wherein the inhalation composition is a nasal spray.
7. The inhalation composition according to claim 1, wherein the inhalation composition comprises about 0.1% w/w to about 40% w/w of nitazoxanide or its derivative thereof.
8. The inhalation composition according to claim 1, wherein the inhalation composition comprises about 0.2% w/w to about 30% w/w of nitazoxanide or its derivative thereof.
9. The inhalation composition according to claim 1, wherein the inhalation composition comprises about 0.5% w/w to about 20% w/w of nitazoxanide or its derivative thereof.
10. The inhalation composition according to claim 1, wherein the inhalation composition has pH of about 4 to about 8.
11. The inhalation composition according to claim 1, wherein the artemisinin or its derivative thereof has a D10 of about 5 to about 30 microns, a D50 of about 20 to about 60 microns, a D90 of about 40 to about 150 microns, a SPAN of not more than about 5.
12. The inhalation composition according to claim 1, wherein the inhalation composition comprises a mass median aerodynamic diameter below about 10 microns and a geometric standard deviation of about 5.
13. The inhalation composition according to claim 1, wherein the inhalation composition exhibits a fine particle fraction between about 10% to about 80%.
14. The inhalation composition according to claim 3, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, 1,1,1,2-tetrafluoroethane propellant (HFA-134a), and ethanol.
15. The inhalation composition according to claim 4, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, Polysorbate 80, disodium EDTA, sodium chloride, citric acid monohydrate and sodium citrate dehydrate, optionally a pH adjusting agent and water.
16. The inhalation composition according to claim 4, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, sulfobutylether-ß-cyclodextrin, optionally a pH adjusting agent and water.
17. The inhalation composition according to claim 4, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, Polysorbate 80, propylene glycol and water.
18. The inhalation composition according to claim 4, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, a lipid, a surfactant, and water.
19. The inhalation composition according to claim 4, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, dipalmitoyl phosphatidylcholine, Polysorbate 80, and water.
20. The inhalation composition according to claim 4, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, dipalmitoyl phosphatidylcholine, Polysorbate 80, and water.
21. The inhalation composition according to claim 5, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, and lactose.
22. The inhalation composition according to claim 6, wherein the inhalation composition comprises nitazoxanide or its derivative thereof, xanthan gum, benzalkonium chloride, sodium chloride, disodium EDTA, phosphate buffer, Polysorbate 80 and water.